Neuronal Glycogen Breakdown Mitigates Tauopathy via Pentose Phosphate Pathway-Mediated Oxidative Stress Reduction

Abstract

Tauopathies encompass a range of neurodegenerative disorders, such as Alzheimer's disease (AD) and frontotemporal dementia (FTD). Unfortunately, current treatment approaches for tauopathies have yielded limited success, underscoring the pressing need for novel therapeutic strategies. We observed distinct signatures of impaired glycogen metabolism in the Drosophila brain of the tauopathy model and the brain of AD patients, indicating a link between tauopathies and glycogen metabolism. We demonstrate that the breakdown of neuronal glycogen by activating glycogen phosphorylase (GlyP) ameliorates the tauopathy phenotypes in flies and induced pluripotent stem cell (iPSC) derived neurons from FTD patients. We observed that glycogen breakdown redirects the glucose flux to the pentose phosphate pathway to alleviate oxidative stress. Our findings uncover a critical role for increased GlyP activity in mediating the neuroprotection benefit of dietary restriction (DR) through the cAMP-mediated protein kinase A (PKA) activation. Our studies identify impaired glycogen metabolism as a key hallmark for tauopathies and offer a promising therapeutic target in tauopathy treatment.

Fig 1.. DR increases lifespan and protects against neurodegeneration in tau flies

a, Lifespan of control (Ctrl) flies, elav-Gal4/+ on AL (red) & DR (blue) diets, show extension on DR. b, Lifespan of flies expressing tau^WT in the neuron shows extension on DR. c, Lifespan of flies expressing tau^R406W in the neurons shows extension on DR. d, Mean lifespans of control, tau^WT and tau^R406W flies are increased on DR over flies raised on the AL diet. This experiment represents the mean values of three independent experiments. e, TUNEL staining of control & tau^R406W fly’s midbrain on AL & DR. Red dots indicate TUNEL-positive nuclei, which are increased in tau^R406W on AL diets and rescued by DR diet. f, Quantification of TUNEL staining show that the number of TUNEL-positive cells per area of the tau^R406W fly brain increases on AL diet and is rescued by DR. Dots represent individual fly brains. See also Figure S1. g, Semi-thick sections of tau^R406W fly brain stained with toluidine blue shows increased vacuoles in tau^R406W, which is rescued by DR. h, Quantification of vacuoles per brain section showing that DR rescues increased vacuoles in tau^R406W. Data in panel D represents 3 independent experiments. An asterisk (*) indicates a significant difference between experimental groups and controls, with the level of significance denoted by the number of asterisks p < 0.05 for *, p < 0.01 for **, p < 0.001 for *** and p < 0.0001 for **** by log-rank test (a, b, and c) or by one-way ANOVA (d, f, and h). Data in bar graphs are presented as mean ± SEM.

Fig. 2. Glycogen metabolism is altered in tauopathy, and glycogen breakdown prevents neurodegeneration in Drosophila and iPSC-derived neurons

a, Venn diagram of the number of proteins upregulated in tau^R406W (red circle) as well as in control on AL diets (blue circle). Dots represent enriched pathways of overlapping proteins, including fatty acid and glycogen metabolism. b, Venn diagram shows numbers of proteins down-regulated in tau^R406W (red circle) and control on AL diets (blue circle). Dots represent enriched pathways of overlapping proteins, including oxidative phosphorylation and glutathione metabolism. c, Venn diagram of the overlapping genes altered in humans and flies. The red circle represents altered protein with a human orthologue, and the blue circle represents altered protein in a human AD patient’s brain. d, Schematic diagram of glycogen metabolism with upregulated proteins marked with red upright arrows. e, Correlation of glycogen phosphorylase and phosphoglucomutase protein abundance with AD diagnosis. Asym AD represents asymptomatic AD f, Images show eye degeneration by overexpression of tau^WT in the eye driven by GMR, a constitutively active stable regulator rescued by GlyP^WT overexpression. Either RNAi or overexpression construct was activated by elav Gal4. g, Quantification of the phenotypic score derived from either RNAi or overexpression flies shows GlyP^WT overexpression rescues the phenotypic score of eye degeneration. h, Quantification of glycogen (in μg/μg of protein) in tau^R406W or control on both AL and DR diets shows increased glycogen in tau^R406W fly brain. The red bars represent flies on the AL diet, and the blue bars represent flies on the DR diet. i, Quantification of glycogen (in μg/μg of protein) of control (tau^R406W expressing mutant GlyP^S15A) and overexpression of GlyP^WT in tau^R406W fly brains show a reduction in GlyP^WT in tau^R406W. j, Increased lifespan of GlyP^WT; tau^R406W (green) compared to control GlyP^S15A; tau^R406W flies(red). k, TUNEL staining of whole mount fly brains of GlyP^S15A; tau^R406W and GlyP^WT; tau^R406W. Red dots represent TUNEL-positive cells reduced in the midbrain of GlyP^WT; tau^R406W flies. l, Quantification of TUNEL staining shows that the number of TUNEL-positive cells per brain area is reduced in GlyP^WT; tau^R406W. Dots represent individual fly brains. m, Images represent glycogen staining with fluorescent 2-NBDG in patient iPSC-derived tau^R406W neurons and isogenic control cells(iso-tau^R406W). n, Quantification of glycogen as fluorescence intensity shows an increase in tau^R406W neurons (red) compared to isogenic control neurons(blue). o, Immunocytochemistry of mitochondria labeled with TOM20 (red) counterstained by DAPI (blue) in iso-tau^R406W and tau^R406W neurons with either control lentiviral transduction or PYGB overexpressing lentivirus. p, Quantification of mitochondrial density normalized with MAP2 area shows that reduced mitochondria in tau^R406W neurons are rescued by PYGB overexpression. Each dot represents an image field from n=3 coverslips per condition for N and P. See also Table 1 in supplementary, Figure S2, supplementary data 1, and supplementary data 2. An asterisk (*) indicates a significant difference between experimental groups and controls, with the level of significance denoted by the number of asterisks p < 0.05 for *, p < 0.01 for **, p < 0.001 for *** and p < 0.0001 for **** by Fisher’s exact (a, b, and c), by one-way ANOVA (e, g, and h), by Two-way ANOVA (p), by Student’s t-test (i, l, and n) or by log-rank test (j). Data in bar graphs are presented as mean ± SEM.

Fig 3.. Glycogen breakdown shunts glucose to the pentose phosphate pathway and reduces oxidative stress

a, Heatmap of significantly (p<0.05) altered metabolites compared between GlyP^S15A; tau^R406W and GlyP^WT; tau^R406W. The green spectrum represents upregulated metabolites, and the red represents downregulated metabolites. b, Quantification of Ribulose 5-phosphate shows its abundance is higher in GlyP^S15A; tau^R406W (red) than control GlyP^WT; tau^R406W (green). c, Quantification of acetyl coA shows a reduction in GlyP^S15A; tau^R406W (red) fly brain compared to GlyP^WT; tau^R406W (green). d, Images show ROS staining by DCFDA in the wholemount brains of GlyP^S15A; tau^R406W, and GlyP^WT; tau^R406W fly with and without 6-AN treatment. e, Quantification of fluorescence intensity of DCFDA staining shows a reduction in GlyP^WT; tau^R406W (green) compared to GlyP^S15A; tau^R406W (red) and increased in 6-AN treatment (green hatched bar). f, Lifespan extension of GlyP^WT; tau^R406W (green) compared to GlyP^S15A; tau^R406W (red) is abrogated with 6-AN treatment (green dashed line). g, Images represent TUNEL staining of wholemount brain of GlyP^S15A; tau^R406W and GlyP^WT; tau^R406W with and without 6-AN treatment. Red dots represent TUNEL-positive cells. h, Quantification shows reduced TUNEL positive cells in GlyP^WT; tau^R406W (green) than GlyP^S15A; tau^R406W (red) and an increase with 6-AN treatment (green checkered bar). i, Schematic diagram shows that glycogen catabolism induces the pentose phosphate pathway and reduces the glycolysis and TCA cycle. Arrows represent altered pathway intermediates or enzyme expression. See also Figure S3, Supplementary Data 3, Supplementary Data 4. An asterisk (*) indicates a significant difference between experimental groups and controls, with the level of significance denoted by the number of asterisks p < 0.05 for *, p < 0.01 for **, p < 0.001 for *** and p < 0.0001 for **** by Student’s t-test (b and c), by one-way ANOVA (e and h) or by log-rank test (f). Data in bar graphs are presented as mean ± SEM.

Fig. 4. DR activates GlyP by activating the cAMP/PKA pathway

a, Schematic diagram of upstream activator of GlyP. b, GlyP activity in brain lysate of control and tau^R406W on DR and with 8-Br-cAMP treatment on AL diet. Throughout the figure, red, blue, and green colors represent AL, DR, and AL + 8-Br-cAMP. c, Relative mRNA expression of the rut gene (normalized with RP49) in tau^R406W is downregulated in AL and rescued in the DR diet. d, Protein abundance of AC is reduced in AD patients. e, cAMP concentration is increased in control and tau^R406W flies on the DR diet compared to the AL diet. f, Western blot of PKA-C1 and actin of brain tissue lysate of control and tau^R406W on AL and DR. g, Normalized densitometric analysis of western blot shows decreased abundance of PKA-C1 of Tau^R406W on AL h, PKA activity increases with tau^R406W reared on DR than AL i, Lifespan of tau^R406W flies reared on AL is extended with 8-Br-cAMP treatment (dash red). j, Images show TUNEL staining of tau^R406W fly brains with and without 8-Br-cAMP treatment. Red dots represent TUNEL-positive cells in the midbrain. k, 8-Br-cAMP treatment decreases TUNEL positive cells in tau^R406W flies. Dots represent individual brains. l, Images show ROS stained with DCFDA in tau^R406W treated with 8-Br-cAMP. m, ROS are reduced in tau^R406W fly brains treated with 8-Br-cAMP. Dots represent individual brains. n, Schematic diagram of the mechanism of DR-mediated neuroprotection and lifespan extension by activation of GlyP via cAMP-mediated PKA activation. An asterisk (*) indicates a significant difference between experimental groups and controls, with the level of significance denoted by the number of asterisks p < 0.05 for *, p < 0.01 for **, p < 0.001 for *** and p < 0.0001 for **** by Student’s t-test (k and m), by one-way ANOVA (b, c, d, e, g, and h) or by log-rank test (i). Data in bar graphs are presented as mean ± SEM.